: Although it is well known that cellular rejection is mediated by alloreactive lymphocytes, several investigators, including our group, have shown that such cells are a rather small proportion of the T cell infiltrate of the allograft. We have therefore postulated that graft-infiltrating lymphocytes must recognize other antigens. Since heat shock protein (hsp)-specific lymphocytes have been shown to participate in several autoimmune diseases and in tumor immunity, we hypothesized that hsp-reactive lymphocytes are involved with allograft rejection. This hypothesis was tested with a rat model of heterotopic MHC-incompatible cardiac allografts (ACI into Lewis), whereby graft-infiltrating lymphocytes and spleen cells were tested in vitro with different recombinant mycobacterial hsp preparations. As expected, allograft lymphocytes showed proliferative responses to irradiated spleen cells from the donor. This proliferation was markedly augmented by hsp65 (3-fold) and hsp70 (5-fold), whereas hsp10 and the protein control ovalbumin had no effect. Proliferation of allograft lymphocytes to hsp in context with syngeneic splenocytes as antigen-presenting cells (APC) was seen primarily if small quantities of IL-2 had been added to the cultures. In contrast, hsp-specific proliferation was never observed with syngraft lymphocytes, even after addition of IL-2. Spleen cells from allograft and syngraft recipients showed hsp augmentation of alloproliferation, but the magnitude was less than that with allograft lymphocytes. Kinetic studies showed that hsp-reactive lymphocytes first appeared in the allograft on day 3 posttransplant. Tacrolimus immunosuppression of transplant rejection prevented the appearance of hsp-reactive lymphocytes in allografts. Culture conditions have been established to generate hsp65-and hsp70-specific T lymphocyte lines and clones from allograft-infiltrating cells. These cultured cells exhibited hsp reactivity only in context with self-APC, and this was augmented by small amounts of IL-2. These data provide strong evidence for the involvement of hsp-reactive lymphocytes in allograft rejection. We propose the concept that during rejection tissue stress induced by alloreactive effector lymphocytes promotes the recruitment and activation of hsp-reactive lymphocytes, especially in the presence of IL-2 released into the allogeneic environment of the transplant. These hsp-reactive T cells may play a role in the immune cascade of the inflammatory process of transplant rejection.